I have tried expressing 3 different proteins in yeast, all are different sizes, 8kDa, 11kDa and 15kDa. However, when I run the expressed proteins on a 4-12% SDS-PAGE they all appear as the same size ~12kDa. I have completed a western blot and the his-tag is present on all 3...Am I looking at 3 different proteins or the same thing?
I've attached the 3 different gene/plasmid constructs, where we have an alpha secretion signal, a his-tag, TEV cleavage site and followed bythe protein of interest. Is there something wrong with these sequences that would cause the same product to be expressed across the 3 different cultures?
Any help, or your best guess would be appreciated. Thank you.
If they were cloned correctly, there is no reason to believe something wrong is happening. But it is indeed weird they appear the same size in an SDS-PAGE. Do you have access to mass spectrometry? An intact analysis should quickly tell you the mass of your expressed proteins.
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